Rescue of ΔF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway

The most prevalent disease-causing mutation of CFTR is the deletion of Phe508 (ΔF508), which leads to defects in conventional Golgi-mediated exocytosis and cell surface expression. We report that ΔF508-CFTR surface expression can be rescued in vitro and in vivo by directing it to an unconventional GRASP-dependent secretion pathway. An integrated molecular and physiological analysis indicates that mechanisms associated with ER stress induce cell surface trafficking of the ER core-glycosylated wild-type and ΔF508-CFTR via the GRASP-dependent pathway. Phosphorylation of a specific site of GRASP and the PDZ-based interaction between GRASP and CFTR are critical for this unconventional surface trafficking. Remarkably, transgenic expression of GRASP in ΔF508-CFTR mice restores CFTR function and rescues mouse survival without apparent toxicity. These findings provide insight into how unconventional protein secretion is activated, and offer a potential therapeutic strategy for the treatment of cystic fibrosis and perhaps diseases stemming from other misfolded proteins.     

Movement of small‐bodied fishes from Lake Michigan into Chicago Area Waterways: Insights from otolith chemistry

The Chicago Area Waterways System (CAWS), an artificial connection between Lake Michigan (LMI) and the Illinois River watershed, has served as a conduit for invasive species and is a potential pathway for viral hemorrhagic septicemia (VHS) to spread from the Great Lakes into the Illinois River drainage. Although some fishes are known to have moved from LMI into the Illinois River via the CAWS, the rate of fish passage from the lake into the CAWS is unclear. Stable carbon isotopic signatures (δ¹³C) in otoliths differ between lake‐ and CAWS‐resident fish and were used as a natural tag to identify individuals that may have moved into the CAWS from LMI. The objectives of this study were to (i) estimate relative frequency of individuals with otolith δ¹³C indicative of prior residency in LMI among small‐bodied (<150 mm total length) VHS‐susceptible fishes collected from several locations in the CAWS, and (ii) to assess differences in relative frequency of probable immigrants from LMI among CAWS locations that differ in distance from the lake. Thirty‐six percent of small‐bodied fishes collected from the CAWS were identified as potential immigrants based on otolith δ¹³C. The percentage of individuals that had otolith δ¹³C suggestive of prior residency in LMI was higher among fish collected from the Calumet River compared to the Chicago River and North Shore Channel and decreased with increasing distance from lock and dam structures at the three entrances to the CAWS from the lake. Movement of small‐bodied fishes from LMI into the CAWS appears to be common and may represent a pathway for VHS‐susceptible species to transport the virus into the CAWS and potentially beyond.     

N‐cadherin/catenin–based costameres in cultured chicken cardiomyocytes

N‐cadherin is a member of the Ca²⁺‐dependent cell adhesion molecules and plays an important role in the assembly of the adherens junction in chicken cardiomyocytes. In addition to being present at the cell‐cell junction, N‐cadherin is associated with costameres in extrajunctional regions. The significance of the N‐cadherin‐associated costameres and whether catenins are components of costameres in chicken cardiomyocytes are not known. In this study, double‐labeling immunofluorescence microscopy was used to determine the extrajunctional distribution of both N‐cadherin and its cytoplasmic associated proteins, α‐ and β‐catenins, and their relationship to myofibrillar Z‐disc α‐actinin. N‐cadherin, α‐, and β‐catenins were all found to be present at the extrajunctional region and, in some cases, were codistributed with myofibrillar α‐actinin exhibiting a periodic staining pattern. Confocal microscopy confirmed that both N‐cadherin and β‐catenin colocalized with peripheral myofibrillar α‐actinin on the dorsal surface of cardiomyocytes as components of the costameres. Intracellular application of antibodies specific for the cytoplasmic portions of N‐cadherin, α‐, and β‐catenin, either by electroporation or microinjection, resulted in myofibril disorganization and disassembly. These results suggest the existence of N‐cadherin/catenin‐based costameres in the dorsal surface of cultured chicken cardiomyocytes in addition to the integrin/vinculin‐based costameres found in the ventral surface and indicate that the former set of costameres is essential for cardiac myofibrillogenesis. J. Cell. Biochem. 75:93–104, 1999. © 1999 Wiley‐Liss, Inc.     

Expression profiles of human endogenous retrovirus (HERV)-K and HERV-R Env proteins in various cancers

The vertebrate genome contains an endogenous retrovirus that has been inherited from the past millions of years. Although approximately 8% of human chromosomal DNA consists of sequences derived from human endogenous retrovirus (HERV) fragments, most of the HERVs are currently inactive and non-infectious due to recombination, deletions, and mutations after insertion into the host genome. Several studies suggested that Human endogenous retroviruses (HERVs) factors are significantly related to certain cancers. However, only limited studies have been conducted to analyze the expression of HERV derived elements at protein levels in certain cancers. Herein, we analyzed the expression profiles of HERV-K envelope (Env) and HERV-R Env proteins in eleven different kinds of cancer tissues. Furthermore, the expression patterns of both protein and correlation with various clinical data in each tissue were analyzed. The expressions of both HERV-K Env and HERV-R Env protein were identified to be significantly high in most of the tumors compared with normal surrounding tissues. Correlations between HERV Env expressions and clinical investigations varied depending on the HERV types and cancers. Overall expression patterns of HERV-K Env and HERV-R Env proteins were different in every individual but a similar pattern of expressions was observed in the same individual. These results demonstrate the expression profiles of HERV-K and HERV-R Env proteins in various cancer tissues and provide a good reference for the association of endogenous retroviral Env proteins in the progression of various cancers. Furthermore, the results elucidate the relationship between HERV-Env expression and the clinical significance of certain cancers.     

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae)

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm‐isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)‐like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria‐colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high‐affinity sucrose‐specific proton‐dependent importer. Plasma‐membrane/tonoplast localization of GeSUT4‐GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant‐microbe interactions.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.